Biosensors and Bioelectronics 72 (2015) 51–55
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Fluorescent sensing of pyrophosphate anion in synovial fluid based on DNA-attached magnetic nanoparticles Li-li Tong a, Zhen-zhen Chen a, Zhong-yao Jiang a, Miao-miao Sun a, Lu Li a, Ju Liu b, Bo Tang a,n a College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, PR China b Laboratory of Microvascular Medicine, Medical Research Center, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 24 February 2015 Received in revised form 20 April 2015 Accepted 27 April 2015 Available online 30 April 2015
In this work, a new fluorescent method for sensitive detection of pyrophosphate anion (P2O74 , PPi) in the synovial fluid was developed using fluorophore labeled single-stranded DNA-attached Fe3O4 NPs. The sensing approach is based on the strong affinity of PPi to Fe3O4 NPs and highly efficient fluorescent quenching ability of Fe3O4 NPs for fluorophore labeled single-stranded DNA. In the presence of PPi, the fluorescence would enhance dramatically due to desorption of fluorophore labeled single-stranded DNA from the surface of Fe3O4 NPs, which allowed the analysis of PPi in a very simple manner. The proposed sensing system allows for the sensitive determination of PPi in the range of 2.0 10 7–4 10 6 M with a detection limit of 76 nM. Importantly, the protocol exhibits excellent selectivity for the determination of PPi over other phosphate-containing compounds. The method was successfully applied to the determination of PPi in the synovial fluid, which suggests our proposed method has great potential for diagnostic purposes. & 2015 Elsevier B.V. All rights reserved.
Keywords: Pyrophosphate anion Fe3O4 nanoparticles Fluorescent sensing Fluorophore labeled single-stranded DNA Synovial fluid
1. Introduction Pyrophosphate anion (P2O74 , PPi) is an important molecule in biological systems because it plays significant roles in several bioenergetics and metabolic processes especially the pathological processes of arthritis (Caswell et al., 1983; Doherty et al., 1996; Johnson and Terkeltaub, 2005; Kulaev et al., 1999; Lawrence et al., 1998; Lipscomb and Sträter, 1996; Mansurova, 1989; Terkeltaub, 2001). Early efforts have suggested that PPi could be used as a potential biomarker for the clinic diagnosis and therapy of arthritis diseases (Doherty et al., 1996; Micheli et al., 1981; Russell et al., 1971; Terkeltaub, 2001). For instance, the high level of PPi in the synovial fluid has been proposed as an index for arthritis, closely relating to the pathogenesis of calcium pyrophosphate dehydrates deposition diseases (CPDD) (Doherty et al., 1996; Terkeltaub, 2001). Therefore, measuring the level of PPi is very import for early stage monitoring of the corresponding health problems. During the past decade, fluorescent chemosensors and colorimetric sensors for PPi have attracted considerable attention due to the intrinsical high sensitivity, simplicity, and ease of operation. A n
Corresponding author. E-mail address: [email protected] (B. Tang).
http://dx.doi.org/10.1016/j.bios.2015.04.087 0956-5663/& 2015 Elsevier B.V. All rights reserved.
number of organic probes have been synthesized for PPi detection base on metal–ion-complex, even imaging studies for intracellular PPi (Bhowmik et al., 2014; Fabbrizzi et al., 2002; Kim et al., 2008; Lee et al., 2004; Lee et al., 2007; O’Neil and Smith, 2006; VillamilRamos and Yatsimirsky, 2011). Some of the probes require elaborate organic design and may not function in aqueous solution, which have limited their practical application. Recently, nanomaterials are interesting for developing biosensors owing to their good optical properties, such as gold nanoparticles, gold nanoclusters, grapheme oxide, magnetic nanoparticles (Chen et al., 2013; Chen et al., 2015; Jung et al., 2010; Li et al., 2012a; Liu et al., 2011; Liu et al., 2012; Saha et al., 2012; Zhang et al., 2011a; Zhang et al., 2011b). Nanomaterials-based chemosensor using metal–ioncomplex were also reported for PPi detection (Oh et al., 2011; C. Zhang et al., 2011a, J.F. Zhang et al., 2011b; Deng et al., 2013; Kim et al., 2013; Li et al,. 2012b; Liu et al., 2013; Yu et al., 2013). For example, Han group reported chemical functionalized-gold nanoparticles as selective colorimetric probes for PPi detection (Kim et al., 2013). This strategy has good sensitivity and selectivity for PPi, but this method requires time-consuming procedure for synthesis of ion-specific ligands, chemical modification and separation. Li et al. (2012a) utilized gold nanoclusters as selective luminescent probes for the detection of PPi. But this method
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suffers from low discrimination capability between PPi and other compounds containing phosphate groups. As a consequence, a great incentive still exists for the development of a new nanomaterials-based fluorescent sensor for the detection of PPi with simplicity, sensitivity and selectivity. In recent years, magnetic nanoparticles (Fe3O4 NPs) have attracted much attention in nanotechnology due to their low costs, superior stability and reusability (Gao et al., 2007). Compared to other nanomaterials, Fe3O4 NPs are advantageous in practical applications because it was readily isolated from sample solutions by an external magnetic field without additional centrifugation or filtration (Beveridge et al., 2011; Ito et al., 2006). Except of these merits, Fe3O4 NPs have been proved to be an efficient fluorescent quencher for boron-dipyrromethene through photo-induced electron transfer from excited states to an unfilled d shell of iron cations on the nanoparticle surface (Yu et al., 2013). On the other hand, previous studies have shown that iron cations on the surface of Fe3O4 NPs coordinates with phosphate-containing compounds through covalent bonding (Lee et al., 2008; Sahoo et al., 2001). Inspired by these excellent properties of Fe3O4 NPs, we reason that Fe3O4 NPs can efficiently quench the fluorescence of fluorophore labeled single-stranded DNA by DNA-attached Fe3O4 NPs, which can be used to develop a new and convenient fluorescence detection method for highly selective and sensitive detection of PPi. Based on the competitive coordination of Fe3O4 NPs between PPi and the fluorophore labeled single-stranded DNA, we established a fluorescent sensing assay for PPi with a DNA-attached Fe3O4 NPs. Under the optimal conditions, the protocol exhibits excellent selectivity for the determination of PPi over other phosphate-containing compounds. The method was successfully used for the detection of PPi in the synovial fluid of arthritis patients, which suggested the present approach had great practicability for diagnostic purposes.
2. Materials and methods 2.1. Chemicals and reagents The ultra-PAGE-purified and MS-verified random oligonucleotides (F*-ssDNA: 5′-FAM-GGAAGGTGTGGAAGG-3’) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Iron(III) chloride hexahydrate (FeCl3 6H2O), iron(II) chloride tetrahydrate (FeCl2 4H2O), ammonium hydroxide were obtained from Beijing Chemical Company (China). 6-Carboxyfluorescein, Sodium pyrophosphate, Adenosine triphosphate (ATP), adenosine 5’-diphosphate (ADP), adenosine 5′-monphosphate (AMP), Baker's yeast inorganic pyrophosphatase (PPase, EC 3.6.1.1) was purchased from Sigma-Aldrich. All chemicals were of at least analytical grade reagents and used without further purification. All solutions were prepared with water purified by a Milli-Q Purification System (Millipore, USA). Unless otherwise noted, all experiments were carried out at room temperature. 2.2. Instrumentation Fluorescence spectra were measured on Edinburgh FLS-920 spectrophotometer (Edinburgh Instruments Ltd., UK) equipped with 450 W Xenon lamp excitation source, using a quartz cell of 1.0 cm path length. The absorption spectra were recorded using a synergy Mx UV visible spectrophotometer (BioTek, USA). DNA concentrations were estimated by using the 260 nm UV absorbance and the corresponding extinction coefficients. Transmission electron microscopy (Hitachi Model H-800 instrument) was used to determine the size of the magnetic nanoparticles.
2.3. Synthesis of magnetic nanoparticles Magnetic nanoparticles were synthesized as reported before (Yu et al., 2010). Briefly, FeCl3 6H2O (2.16 g) and FeCl2 4H2O (0.8 g) were dissolved in 20 mL of deionized water. The resulting solution was deoxygenated by bubbling with nitrogen gas for 10 min and then heated to 80 °C with stirring in a round bottom flask. Aqueous NH4OH (8 M, 10 mL) was added rapidly to the heated solution, which was stirred for 1 h. After cooling to room temperature, the formed Fe3O4 NPs were rinsed four times with deionized water and then resuspended in N2 saturated water (100 mL). The concentration of Fe3O4 NPs was estimated to be about 10 mg/mL. The size of the synthetic nanoparticles was about 18.0 nm as confirmed by TEM image (Fig. S1, Supporting information). 2.4. Preparation of Fe3O4 NPs/F*-ssDNA complex materials Firstly, the fluorophore-FAM labeled single-stranded DNA (F*ssDNA) solution (in 50 mM Tris–HCl buffer, pH: 7.2) was heated at 90 °C for 10 min and gradually cooled to room temperature. Then the desired concentration of Fe3O4 NPs and above mentioned F*ssDNA (both diluted with 50 mM Tris–HCl buffer containing 50 mM NaCl) were mixed and incubated for 10 min to allow the formation of Fe3O4 NPs/ F*-ssDNA complex. In all cases, samples were excited at 488 nm, and the emission was monitored at 518 nm. An optimized concentration of Fe3O4 NPs was achieved by monitoring fluorescence change of Fe3O4 NPs/ F*-ssDNA complex solution. 2.5. Fluorescence detection of pyrophosphate anion PPi with varying concentrations prepared in distilled water were added into as-prepared mixture solution of Fe3O4 NPs/F*ssDNA complex (Fe3O4 NPs 10 μg/mL, F*-ssDNA 50 nM). The resulting mixtures were allowed to react for 30 min at room temperature and then used for fluorescence measurements. The final concentrations of PPi in the resulting mixtures were 0.20 μM, 0.30 μM, 0.50 μM, 1.0 μM, 2.0 μM, 3.0 μM and 4.0 μM. The measuring processing was the same as above. 2.6. Fluorescence sensing of PPi in synovial fluids of arthritis patients The synovial fluids of arthritis patients were obtained from the local hospital. Prior to the fluorescence sensing, the synovial fluids were purified by perfusing the fluid through a microdialysis probe. Then these samples were diluted with 50 mM Tris–HCl buffer (pH: 7.2) in order to be consistent with the dynamic range of our method. Aliquots were mixed with the Fe3O4 NPs/F*-ssDNA complex directly, and same amount of aliquots were pretreated with inorganic pyrophosphatase (PPase) as a PPi-blocking compound (Altman et al., 1973; Russell et al., 1971) before reaction with the Fe3O4 NPs/F*-ssDNA complex and subsequent fluorescence measurement. PPi of known concentrations were added to the samples for recovery studies and the PPi concentrations were determined by the standard addition method.
3. Results and discussion 3.1. Principle of pyrophosphate anion detection The general scheme of the new turn-on fluorescence sensor for PPi is illustrated in Fig. 1. Initially, the fluorophore-FAM labeled single-stranded DNA (F*-ssDNA) showed strong fluorescence in aqueous solution. In the presence of in Fe3O4 NPs, its fluorescence
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PPi and F*-ssDNA. PPi can effectively bind to the surface of Fe3O4 NPs, resulting in release of F*-ssDNA from the surface of the Fe3O4 NPs, thus the fluorescence could be turned on. Overall, these observations demonstrate that Fe3O4 NPs/F*-ssDNA complex can be utilized as a novel and simple probe for the analysis of PPi. 3.2. Optimization of detection conditions
Fig. 1. Schematic illustration of the fluorescent assay for pyrophosphate anion based on the fact that pyrophosphate can compete with F*-ssDNA for binding sites on the Fe3O4 NPs surface.
intensity displays remarkable decrease due to the formation of the Fe3O4 NPs/F*-ssDNA complex. After addition of PPi to the Fe3O4 NPs/F*-ssDNA complex, the iron cations on the surface of Fe3O4 NPs were sequestrated and the more stable Fe3O4 NPs/PPi complex was formed, thus releasing the F*-ssDNA and giving out strong fluorescence. To confirm the feasibility of this strategy, Fe3O4 NPs were firstly proved to efficiently quench the fluorescence of fluorophore labeled single-stranded DNA, and the results were illustrated in Fig. 2. In the absence of Fe3O4 NPs, the FAM labeled single-stranded DNA in the random coil state showed strong fluorescence. After incubation with Fe3O4 NPs, a noteworthy decrease in F*ssDNA fluorescence was observed when collecting Fe3O4 NPs by an external magnetic field (Fig. 2 curve a, b). It was very interesting to note that Fe3O4 NPs cannot quench the fluorescence of free FAM solely, because we found that the free FAM fluorescence remained nearly constant after removing Fe3O4 NPs (Fig. S2) when utilized the same procedure as mentioned above. This observation clearly reflect the importance of DNA, it may act as important bridge between Fe3O4 NPs and fluorophores-FAM. We reasoned that F*ssDNA was adsorbed on the surface of the Fe3O4 NPs mainly through the stronger interaction between iron cations and guanine N-7 of DNA bases, as well as the interaction between iron cations and the backbone phosphate group (Lee et al., 2008; Ouameur et al., 2005; Sahoo et al., 2001). As a result of this shortened distance between F*-ssDNA and Fe3O4 NPs, the fluorescence of F*ssDNA could be quenched by Fe3O4 NPs effectively photo-induced electron transfer. We next monitored the fluorescence change of Fe3O4 NPs/F*ssDNA in the presence of PPi (Fig. 2 curve c). The results showed after adding PPi to Fe3O4 NPs/F*-ssDNA complex, the fluorescence enhancement was significant. This phenomenon may be attributed to the competitive coordination interaction of Fe3O4 NPs between
Fig. 2. The fluorescence emission spectra of F*-ssDNA (a) and F*-ssDNA/ Fe3O4 NPs (b), PPi þFe3O4 NPs/F*-ssDNA (c). The final concentrations of F*-ssDNA, Fe3O4 NPs, PPi are 50 nM, 10 μg/mL and 2.0 μM, respectively.
Several detection conditions that may affect the fluorescence assay of the target PPi were optimized, such as the concentration of Fe3O4 NPs, and incubation time of PPi. Fluorescence titration experiment was first performed in the presence of F*-ssDNA at a fixed concentration of 50 nM. The fluorescence intensity of F*ssDNA decreased significantly with increasing Fe3O4 NPs concentration until the concentration reached 20 μg/mL (Fig. 3A). In order to obtain an optimum detection sensitivity and a wide detection range, a concentration of 10 μg/mL (approximately 75% quenching of initial fluorescence) was selected for the following experiments. Then the influence of time on the fluorescence intensity changes of the Fe3O4 NPs/ F*-ssDNA complex with PPi incubation was investigated (Fig. 3B). The fluorescence intensity increased rapidly within 20 min. After 30 min, it remained nearly constant. Therefore, an optimal incubation time of 30 min was selected for PPi detection. 3.3. Sensitivity and selectivity for pyrophosphate anion To explore the feasibility of this Fe3O4 NPs/F*-ssDNA complex sensor approach for quantitative analysis, under the optimal conditions, fluorescence spectroscopy was used to evaluate the response of the sensors toward PPi. As shown in Fig. 4A, the fluorescence responses of the system gradually raised with the increase of PPi concentration. A good linear relationship between fluorescent enhancement (F F0, F and F0 are fluorescence intensities in the in presence and absence of PPi, respectively) and pyrophosphate concentrations was obtained over the range from 0.20 μM to 4.0 μM (R2 ¼ 0.9906, Fig. 4B). The limit of detection (LOD) as low as 76 nM was achieved according to the assumption of LOD equal to 3 SD/S, where SD is the standard error of the intercept and S is the slope of the calibration curve. This value is lower or comparable to those reported fluorescent sensors for PPi. Discrimination of trace amounts of PPi from a large excess of other anions and other compounds containing phosphate groups is a critical issue for PPi detection. We examined the selectivity of the Fe3O4 NPs/F*-ssDNA sensor for PPi by comparison of its response to PPi with potentially interfering substances that possibly exist in synovial fluid at physiological concentration ratios. As shown in Fig. 5, the response signal to PPi was strikingly larger than other tested species including anions and nucleotides. This excellent selectivity may be attributed to two factors: the structure and charge density of PPi; and the steric hindrance effect of the Fe3O4 NPs/F*-ssDNA complex. The total anionic charge density on the four O–P oxygen atoms of PPi is larger than other phosphatecontaining compounds, which offers much stronger binding affinity to Fe3O4 NPs (Su et al., 2013). On the other hand, the steric hindrance makes PPi easier to reach the Fe3O4 NPs/F*-ssDNA complex than other nucleotides. To further confirm this, we studied the effect of different chemical addition order on fluorescence change (Fig. S3). When compounds containing phosphate groups such as PPi, ATP, ADP, AMP, PO43 were first added to aqueous Fe3O4 NPs followed by the addition of F*-ssDNA, stronger fluorescence were observed compared with the order that we described above for PPi determination (that is, compounds containing phosphate groups was added into the pre-mixed Fe3O4 NPs/ F*-ssDNA solultion). Besides, it could be found that PPi gave out the highest fluorescence and the fluorescence change was the least
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Fig. 3. (A) The relationship between the fluorescence intensity of F*-ssDNA and the concentration of Fe3O4 NPs (from 0 to 25 μg/mL). The concentration of F*-ssDNA, is 50 nM. (B) Time-dependent fluorescence responses of the Fe3O4 NPs /F*-ssDNA complex in presence of PPi. The final concentrations of F*-ssDNA, Fe3O4 NPs, PPi are 50 nM, 10 μg/mL and 2.0 μM, respectively. The error bar represents the standard deviation of three measurements.
among these compounds. This phenomena demonstrated the largest binding affinity between Fe3O4 NPs and PPi, hence the optimum competitive coordination among PPi, F*-ssDNA and Fe3O4 NPs, which made excellent selectivity of PPi possible with Fe3O4 NPs/F*-ssDNA complex. 3.4. Detecting pyrophosphate anion in synovial fluid samples To evaluate our proposed method for practical applications, the new sensor system was investigated to detect PPi in synovial fluid. As illustrated by the data plotted in Fig. S4, a high fluorescence signal was obtained when synovial fluid was present in the Fe3O4 NPs/F*-ssDNA complex, indicating that the synovial fluid contained certain levels of PPi. In contrast, PPase pretreatment of samples resulted in no distinct increase of the fluorescence intensity because PPase specifically catalyzes the hydrolysis of PPi. This observation shows that fluorescence changes in the assay system were entirely caused by PPi present in the diluted synovial fluid samples. The concentrations of PPi present in the tested synovial fluid samples were determined by employing the standard addition method (Fig. S5). The results were listed in Table 1, which were in good agreement with those obtained in previous studies (Deng et al., 2013). The good recoveries of known amount of PPi in
Fig. 5. The relative fluorescence intensity of Fe3O4 NPs /F*-ssDNA complex at λem ¼ 518 nm in the presence of anion and nucleotides, where F and F0 are fluorescence intensities of Fe3O4 NPs /F*-ssDNA complex in the presence and absence of different anion and nucleotides. The final concentrations of F*-ssDNA, Fe3O4 NPs, PPi, anion and nucleotides are 50 nM, 10 μg/mL, 2.0 μM, 20.0 μM and 2.0 μM respectively. The error bar represents the standard deviation of three measurements.
Fig. 4. (A) Fluorescence emission spectra of Fe3O4 NPs/F*-ssDNA complex in the presence of increasing concentration of PPi (from bottom to top: 0, 0.20, 0.30, 0.50, 1.0, 2.0, 3.0 and 4.0 μM). (B) The relationship between fluorescence intensity change at λem ¼518 nm and concentration of PPi. The final concentrations of F*-ssDNA and Fe3O4 NPs are 50 nM and 10 μg/mL respectively. The error bar represents the standard deviation of three measurements.
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References
Table 1 Determination of pyrophosphate in synovial fluid. Determined PPi Sample PPi in synovial fluid in diluted synovial fluid (μM) (μM)
Added Measured PPi (μM) PPi (μM)
Recovery (%)
1
24.5
0.245
2
23.8
0.475
3
18.6
1.026
0.50 2.00 0.50 2.00 0.50 2.00
106 96.5 102 97.6 102 99.0
0.53 1.93 0.51 1.95 0.51 1.98
the synovial fluid samples definitely demonstrated the accuracy and reliability of the present method for PPi determination in practical applications.
4. Conclusion In summary, we have demonstrated Fe3O4 NPs can efficiently quench the fluorescence of fluorophore labeled sigle-stranded DNA by DNA-attched Fe3O4 NPs and developed a new, simple and turn-on fluorescent method for highly selective and sensitive detection of PPi. This detection strategy is based on the competitive coordination interaction of Fe3O4 NPs between PPi and the fluorophore labeled single-stranded DNA. The system is simple in design and convenient in operation. In addition, the detection limit of this method is lower than or at least comparable to the previous fluorescence method. Importantly, the protocol exhibits excellent selectivity for the determination of PPi over other phosphatecontaining compounds at their normal concentration level in synovial fluid. The method was successfully used for the detection of PPi in synovial fluid, which suggested the present approach had great practicability for diagnosis and therapy of arthritic disease.
Acknowledgment This work was supported by 973 Program (2013CB933800), National Natural Science Foundation of China (21227005, 21390411, 91313302, and 21105057), Promotive Research Fund for Excellent Young and Middle-aged Scientist of Shandong Province (BS2013SW002) and Program for changjiang Scholars and Innovative Research Team in University.
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.04.087.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Fluorescent sensing of pyrophosphate anion in synovial fluid based on DNA-attached magnetic nanoparticles Li-li Tong a, Zhen-zhen Chen a, Zhong-yao Jiang a, Miao-miao Sun a, Lu Li a, Ju Liu b, Bo Tang a,n a College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, PR China b Laboratory of Microvascular Medicine, Medical Research Center, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 24 February 2015 Received in revised form 20 April 2015 Accepted 27 April 2015 Available online 30 April 2015
In this work, a new fluorescent method for sensitive detection of pyrophosphate anion (P2O74 , PPi) in the synovial fluid was developed using fluorophore labeled single-stranded DNA-attached Fe3O4 NPs. The sensing approach is based on the strong affinity of PPi to Fe3O4 NPs and highly efficient fluorescent quenching ability of Fe3O4 NPs for fluorophore labeled single-stranded DNA. In the presence of PPi, the fluorescence would enhance dramatically due to desorption of fluorophore labeled single-stranded DNA from the surface of Fe3O4 NPs, which allowed the analysis of PPi in a very simple manner. The proposed sensing system allows for the sensitive determination of PPi in the range of 2.0 10 7–4 10 6 M with a detection limit of 76 nM. Importantly, the protocol exhibits excellent selectivity for the determination of PPi over other phosphate-containing compounds. The method was successfully applied to the determination of PPi in the synovial fluid, which suggests our proposed method has great potential for diagnostic purposes. & 2015 Elsevier B.V. All rights reserved.
Keywords: Pyrophosphate anion Fe3O4 nanoparticles Fluorescent sensing Fluorophore labeled single-stranded DNA Synovial fluid
1. Introduction Pyrophosphate anion (P2O74 , PPi) is an important molecule in biological systems because it plays significant roles in several bioenergetics and metabolic processes especially the pathological processes of arthritis (Caswell et al., 1983; Doherty et al., 1996; Johnson and Terkeltaub, 2005; Kulaev et al., 1999; Lawrence et al., 1998; Lipscomb and Sträter, 1996; Mansurova, 1989; Terkeltaub, 2001). Early efforts have suggested that PPi could be used as a potential biomarker for the clinic diagnosis and therapy of arthritis diseases (Doherty et al., 1996; Micheli et al., 1981; Russell et al., 1971; Terkeltaub, 2001). For instance, the high level of PPi in the synovial fluid has been proposed as an index for arthritis, closely relating to the pathogenesis of calcium pyrophosphate dehydrates deposition diseases (CPDD) (Doherty et al., 1996; Terkeltaub, 2001). Therefore, measuring the level of PPi is very import for early stage monitoring of the corresponding health problems. During the past decade, fluorescent chemosensors and colorimetric sensors for PPi have attracted considerable attention due to the intrinsical high sensitivity, simplicity, and ease of operation. A n
Corresponding author. E-mail address: [email protected] (B. Tang).
http://dx.doi.org/10.1016/j.bios.2015.04.087 0956-5663/& 2015 Elsevier B.V. All rights reserved.
number of organic probes have been synthesized for PPi detection base on metal–ion-complex, even imaging studies for intracellular PPi (Bhowmik et al., 2014; Fabbrizzi et al., 2002; Kim et al., 2008; Lee et al., 2004; Lee et al., 2007; O’Neil and Smith, 2006; VillamilRamos and Yatsimirsky, 2011). Some of the probes require elaborate organic design and may not function in aqueous solution, which have limited their practical application. Recently, nanomaterials are interesting for developing biosensors owing to their good optical properties, such as gold nanoparticles, gold nanoclusters, grapheme oxide, magnetic nanoparticles (Chen et al., 2013; Chen et al., 2015; Jung et al., 2010; Li et al., 2012a; Liu et al., 2011; Liu et al., 2012; Saha et al., 2012; Zhang et al., 2011a; Zhang et al., 2011b). Nanomaterials-based chemosensor using metal–ioncomplex were also reported for PPi detection (Oh et al., 2011; C. Zhang et al., 2011a, J.F. Zhang et al., 2011b; Deng et al., 2013; Kim et al., 2013; Li et al,. 2012b; Liu et al., 2013; Yu et al., 2013). For example, Han group reported chemical functionalized-gold nanoparticles as selective colorimetric probes for PPi detection (Kim et al., 2013). This strategy has good sensitivity and selectivity for PPi, but this method requires time-consuming procedure for synthesis of ion-specific ligands, chemical modification and separation. Li et al. (2012a) utilized gold nanoclusters as selective luminescent probes for the detection of PPi. But this method
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suffers from low discrimination capability between PPi and other compounds containing phosphate groups. As a consequence, a great incentive still exists for the development of a new nanomaterials-based fluorescent sensor for the detection of PPi with simplicity, sensitivity and selectivity. In recent years, magnetic nanoparticles (Fe3O4 NPs) have attracted much attention in nanotechnology due to their low costs, superior stability and reusability (Gao et al., 2007). Compared to other nanomaterials, Fe3O4 NPs are advantageous in practical applications because it was readily isolated from sample solutions by an external magnetic field without additional centrifugation or filtration (Beveridge et al., 2011; Ito et al., 2006). Except of these merits, Fe3O4 NPs have been proved to be an efficient fluorescent quencher for boron-dipyrromethene through photo-induced electron transfer from excited states to an unfilled d shell of iron cations on the nanoparticle surface (Yu et al., 2013). On the other hand, previous studies have shown that iron cations on the surface of Fe3O4 NPs coordinates with phosphate-containing compounds through covalent bonding (Lee et al., 2008; Sahoo et al., 2001). Inspired by these excellent properties of Fe3O4 NPs, we reason that Fe3O4 NPs can efficiently quench the fluorescence of fluorophore labeled single-stranded DNA by DNA-attached Fe3O4 NPs, which can be used to develop a new and convenient fluorescence detection method for highly selective and sensitive detection of PPi. Based on the competitive coordination of Fe3O4 NPs between PPi and the fluorophore labeled single-stranded DNA, we established a fluorescent sensing assay for PPi with a DNA-attached Fe3O4 NPs. Under the optimal conditions, the protocol exhibits excellent selectivity for the determination of PPi over other phosphate-containing compounds. The method was successfully used for the detection of PPi in the synovial fluid of arthritis patients, which suggested the present approach had great practicability for diagnostic purposes.
2. Materials and methods 2.1. Chemicals and reagents The ultra-PAGE-purified and MS-verified random oligonucleotides (F*-ssDNA: 5′-FAM-GGAAGGTGTGGAAGG-3’) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Iron(III) chloride hexahydrate (FeCl3 6H2O), iron(II) chloride tetrahydrate (FeCl2 4H2O), ammonium hydroxide were obtained from Beijing Chemical Company (China). 6-Carboxyfluorescein, Sodium pyrophosphate, Adenosine triphosphate (ATP), adenosine 5’-diphosphate (ADP), adenosine 5′-monphosphate (AMP), Baker's yeast inorganic pyrophosphatase (PPase, EC 3.6.1.1) was purchased from Sigma-Aldrich. All chemicals were of at least analytical grade reagents and used without further purification. All solutions were prepared with water purified by a Milli-Q Purification System (Millipore, USA). Unless otherwise noted, all experiments were carried out at room temperature. 2.2. Instrumentation Fluorescence spectra were measured on Edinburgh FLS-920 spectrophotometer (Edinburgh Instruments Ltd., UK) equipped with 450 W Xenon lamp excitation source, using a quartz cell of 1.0 cm path length. The absorption spectra were recorded using a synergy Mx UV visible spectrophotometer (BioTek, USA). DNA concentrations were estimated by using the 260 nm UV absorbance and the corresponding extinction coefficients. Transmission electron microscopy (Hitachi Model H-800 instrument) was used to determine the size of the magnetic nanoparticles.
2.3. Synthesis of magnetic nanoparticles Magnetic nanoparticles were synthesized as reported before (Yu et al., 2010). Briefly, FeCl3 6H2O (2.16 g) and FeCl2 4H2O (0.8 g) were dissolved in 20 mL of deionized water. The resulting solution was deoxygenated by bubbling with nitrogen gas for 10 min and then heated to 80 °C with stirring in a round bottom flask. Aqueous NH4OH (8 M, 10 mL) was added rapidly to the heated solution, which was stirred for 1 h. After cooling to room temperature, the formed Fe3O4 NPs were rinsed four times with deionized water and then resuspended in N2 saturated water (100 mL). The concentration of Fe3O4 NPs was estimated to be about 10 mg/mL. The size of the synthetic nanoparticles was about 18.0 nm as confirmed by TEM image (Fig. S1, Supporting information). 2.4. Preparation of Fe3O4 NPs/F*-ssDNA complex materials Firstly, the fluorophore-FAM labeled single-stranded DNA (F*ssDNA) solution (in 50 mM Tris–HCl buffer, pH: 7.2) was heated at 90 °C for 10 min and gradually cooled to room temperature. Then the desired concentration of Fe3O4 NPs and above mentioned F*ssDNA (both diluted with 50 mM Tris–HCl buffer containing 50 mM NaCl) were mixed and incubated for 10 min to allow the formation of Fe3O4 NPs/ F*-ssDNA complex. In all cases, samples were excited at 488 nm, and the emission was monitored at 518 nm. An optimized concentration of Fe3O4 NPs was achieved by monitoring fluorescence change of Fe3O4 NPs/ F*-ssDNA complex solution. 2.5. Fluorescence detection of pyrophosphate anion PPi with varying concentrations prepared in distilled water were added into as-prepared mixture solution of Fe3O4 NPs/F*ssDNA complex (Fe3O4 NPs 10 μg/mL, F*-ssDNA 50 nM). The resulting mixtures were allowed to react for 30 min at room temperature and then used for fluorescence measurements. The final concentrations of PPi in the resulting mixtures were 0.20 μM, 0.30 μM, 0.50 μM, 1.0 μM, 2.0 μM, 3.0 μM and 4.0 μM. The measuring processing was the same as above. 2.6. Fluorescence sensing of PPi in synovial fluids of arthritis patients The synovial fluids of arthritis patients were obtained from the local hospital. Prior to the fluorescence sensing, the synovial fluids were purified by perfusing the fluid through a microdialysis probe. Then these samples were diluted with 50 mM Tris–HCl buffer (pH: 7.2) in order to be consistent with the dynamic range of our method. Aliquots were mixed with the Fe3O4 NPs/F*-ssDNA complex directly, and same amount of aliquots were pretreated with inorganic pyrophosphatase (PPase) as a PPi-blocking compound (Altman et al., 1973; Russell et al., 1971) before reaction with the Fe3O4 NPs/F*-ssDNA complex and subsequent fluorescence measurement. PPi of known concentrations were added to the samples for recovery studies and the PPi concentrations were determined by the standard addition method.
3. Results and discussion 3.1. Principle of pyrophosphate anion detection The general scheme of the new turn-on fluorescence sensor for PPi is illustrated in Fig. 1. Initially, the fluorophore-FAM labeled single-stranded DNA (F*-ssDNA) showed strong fluorescence in aqueous solution. In the presence of in Fe3O4 NPs, its fluorescence
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PPi and F*-ssDNA. PPi can effectively bind to the surface of Fe3O4 NPs, resulting in release of F*-ssDNA from the surface of the Fe3O4 NPs, thus the fluorescence could be turned on. Overall, these observations demonstrate that Fe3O4 NPs/F*-ssDNA complex can be utilized as a novel and simple probe for the analysis of PPi. 3.2. Optimization of detection conditions
Fig. 1. Schematic illustration of the fluorescent assay for pyrophosphate anion based on the fact that pyrophosphate can compete with F*-ssDNA for binding sites on the Fe3O4 NPs surface.
intensity displays remarkable decrease due to the formation of the Fe3O4 NPs/F*-ssDNA complex. After addition of PPi to the Fe3O4 NPs/F*-ssDNA complex, the iron cations on the surface of Fe3O4 NPs were sequestrated and the more stable Fe3O4 NPs/PPi complex was formed, thus releasing the F*-ssDNA and giving out strong fluorescence. To confirm the feasibility of this strategy, Fe3O4 NPs were firstly proved to efficiently quench the fluorescence of fluorophore labeled single-stranded DNA, and the results were illustrated in Fig. 2. In the absence of Fe3O4 NPs, the FAM labeled single-stranded DNA in the random coil state showed strong fluorescence. After incubation with Fe3O4 NPs, a noteworthy decrease in F*ssDNA fluorescence was observed when collecting Fe3O4 NPs by an external magnetic field (Fig. 2 curve a, b). It was very interesting to note that Fe3O4 NPs cannot quench the fluorescence of free FAM solely, because we found that the free FAM fluorescence remained nearly constant after removing Fe3O4 NPs (Fig. S2) when utilized the same procedure as mentioned above. This observation clearly reflect the importance of DNA, it may act as important bridge between Fe3O4 NPs and fluorophores-FAM. We reasoned that F*ssDNA was adsorbed on the surface of the Fe3O4 NPs mainly through the stronger interaction between iron cations and guanine N-7 of DNA bases, as well as the interaction between iron cations and the backbone phosphate group (Lee et al., 2008; Ouameur et al., 2005; Sahoo et al., 2001). As a result of this shortened distance between F*-ssDNA and Fe3O4 NPs, the fluorescence of F*ssDNA could be quenched by Fe3O4 NPs effectively photo-induced electron transfer. We next monitored the fluorescence change of Fe3O4 NPs/F*ssDNA in the presence of PPi (Fig. 2 curve c). The results showed after adding PPi to Fe3O4 NPs/F*-ssDNA complex, the fluorescence enhancement was significant. This phenomenon may be attributed to the competitive coordination interaction of Fe3O4 NPs between
Fig. 2. The fluorescence emission spectra of F*-ssDNA (a) and F*-ssDNA/ Fe3O4 NPs (b), PPi þFe3O4 NPs/F*-ssDNA (c). The final concentrations of F*-ssDNA, Fe3O4 NPs, PPi are 50 nM, 10 μg/mL and 2.0 μM, respectively.
Several detection conditions that may affect the fluorescence assay of the target PPi were optimized, such as the concentration of Fe3O4 NPs, and incubation time of PPi. Fluorescence titration experiment was first performed in the presence of F*-ssDNA at a fixed concentration of 50 nM. The fluorescence intensity of F*ssDNA decreased significantly with increasing Fe3O4 NPs concentration until the concentration reached 20 μg/mL (Fig. 3A). In order to obtain an optimum detection sensitivity and a wide detection range, a concentration of 10 μg/mL (approximately 75% quenching of initial fluorescence) was selected for the following experiments. Then the influence of time on the fluorescence intensity changes of the Fe3O4 NPs/ F*-ssDNA complex with PPi incubation was investigated (Fig. 3B). The fluorescence intensity increased rapidly within 20 min. After 30 min, it remained nearly constant. Therefore, an optimal incubation time of 30 min was selected for PPi detection. 3.3. Sensitivity and selectivity for pyrophosphate anion To explore the feasibility of this Fe3O4 NPs/F*-ssDNA complex sensor approach for quantitative analysis, under the optimal conditions, fluorescence spectroscopy was used to evaluate the response of the sensors toward PPi. As shown in Fig. 4A, the fluorescence responses of the system gradually raised with the increase of PPi concentration. A good linear relationship between fluorescent enhancement (F F0, F and F0 are fluorescence intensities in the in presence and absence of PPi, respectively) and pyrophosphate concentrations was obtained over the range from 0.20 μM to 4.0 μM (R2 ¼ 0.9906, Fig. 4B). The limit of detection (LOD) as low as 76 nM was achieved according to the assumption of LOD equal to 3 SD/S, where SD is the standard error of the intercept and S is the slope of the calibration curve. This value is lower or comparable to those reported fluorescent sensors for PPi. Discrimination of trace amounts of PPi from a large excess of other anions and other compounds containing phosphate groups is a critical issue for PPi detection. We examined the selectivity of the Fe3O4 NPs/F*-ssDNA sensor for PPi by comparison of its response to PPi with potentially interfering substances that possibly exist in synovial fluid at physiological concentration ratios. As shown in Fig. 5, the response signal to PPi was strikingly larger than other tested species including anions and nucleotides. This excellent selectivity may be attributed to two factors: the structure and charge density of PPi; and the steric hindrance effect of the Fe3O4 NPs/F*-ssDNA complex. The total anionic charge density on the four O–P oxygen atoms of PPi is larger than other phosphatecontaining compounds, which offers much stronger binding affinity to Fe3O4 NPs (Su et al., 2013). On the other hand, the steric hindrance makes PPi easier to reach the Fe3O4 NPs/F*-ssDNA complex than other nucleotides. To further confirm this, we studied the effect of different chemical addition order on fluorescence change (Fig. S3). When compounds containing phosphate groups such as PPi, ATP, ADP, AMP, PO43 were first added to aqueous Fe3O4 NPs followed by the addition of F*-ssDNA, stronger fluorescence were observed compared with the order that we described above for PPi determination (that is, compounds containing phosphate groups was added into the pre-mixed Fe3O4 NPs/ F*-ssDNA solultion). Besides, it could be found that PPi gave out the highest fluorescence and the fluorescence change was the least
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Fig. 3. (A) The relationship between the fluorescence intensity of F*-ssDNA and the concentration of Fe3O4 NPs (from 0 to 25 μg/mL). The concentration of F*-ssDNA, is 50 nM. (B) Time-dependent fluorescence responses of the Fe3O4 NPs /F*-ssDNA complex in presence of PPi. The final concentrations of F*-ssDNA, Fe3O4 NPs, PPi are 50 nM, 10 μg/mL and 2.0 μM, respectively. The error bar represents the standard deviation of three measurements.
among these compounds. This phenomena demonstrated the largest binding affinity between Fe3O4 NPs and PPi, hence the optimum competitive coordination among PPi, F*-ssDNA and Fe3O4 NPs, which made excellent selectivity of PPi possible with Fe3O4 NPs/F*-ssDNA complex. 3.4. Detecting pyrophosphate anion in synovial fluid samples To evaluate our proposed method for practical applications, the new sensor system was investigated to detect PPi in synovial fluid. As illustrated by the data plotted in Fig. S4, a high fluorescence signal was obtained when synovial fluid was present in the Fe3O4 NPs/F*-ssDNA complex, indicating that the synovial fluid contained certain levels of PPi. In contrast, PPase pretreatment of samples resulted in no distinct increase of the fluorescence intensity because PPase specifically catalyzes the hydrolysis of PPi. This observation shows that fluorescence changes in the assay system were entirely caused by PPi present in the diluted synovial fluid samples. The concentrations of PPi present in the tested synovial fluid samples were determined by employing the standard addition method (Fig. S5). The results were listed in Table 1, which were in good agreement with those obtained in previous studies (Deng et al., 2013). The good recoveries of known amount of PPi in
Fig. 5. The relative fluorescence intensity of Fe3O4 NPs /F*-ssDNA complex at λem ¼ 518 nm in the presence of anion and nucleotides, where F and F0 are fluorescence intensities of Fe3O4 NPs /F*-ssDNA complex in the presence and absence of different anion and nucleotides. The final concentrations of F*-ssDNA, Fe3O4 NPs, PPi, anion and nucleotides are 50 nM, 10 μg/mL, 2.0 μM, 20.0 μM and 2.0 μM respectively. The error bar represents the standard deviation of three measurements.
Fig. 4. (A) Fluorescence emission spectra of Fe3O4 NPs/F*-ssDNA complex in the presence of increasing concentration of PPi (from bottom to top: 0, 0.20, 0.30, 0.50, 1.0, 2.0, 3.0 and 4.0 μM). (B) The relationship between fluorescence intensity change at λem ¼518 nm and concentration of PPi. The final concentrations of F*-ssDNA and Fe3O4 NPs are 50 nM and 10 μg/mL respectively. The error bar represents the standard deviation of three measurements.
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References
Table 1 Determination of pyrophosphate in synovial fluid. Determined PPi Sample PPi in synovial fluid in diluted synovial fluid (μM) (μM)
Added Measured PPi (μM) PPi (μM)
Recovery (%)
1
24.5
0.245
2
23.8
0.475
3
18.6
1.026
0.50 2.00 0.50 2.00 0.50 2.00
106 96.5 102 97.6 102 99.0
0.53 1.93 0.51 1.95 0.51 1.98
the synovial fluid samples definitely demonstrated the accuracy and reliability of the present method for PPi determination in practical applications.
4. Conclusion In summary, we have demonstrated Fe3O4 NPs can efficiently quench the fluorescence of fluorophore labeled sigle-stranded DNA by DNA-attched Fe3O4 NPs and developed a new, simple and turn-on fluorescent method for highly selective and sensitive detection of PPi. This detection strategy is based on the competitive coordination interaction of Fe3O4 NPs between PPi and the fluorophore labeled single-stranded DNA. The system is simple in design and convenient in operation. In addition, the detection limit of this method is lower than or at least comparable to the previous fluorescence method. Importantly, the protocol exhibits excellent selectivity for the determination of PPi over other phosphatecontaining compounds at their normal concentration level in synovial fluid. The method was successfully used for the detection of PPi in synovial fluid, which suggested the present approach had great practicability for diagnosis and therapy of arthritic disease.
Acknowledgment This work was supported by 973 Program (2013CB933800), National Natural Science Foundation of China (21227005, 21390411, 91313302, and 21105057), Promotive Research Fund for Excellent Young and Middle-aged Scientist of Shandong Province (BS2013SW002) and Program for changjiang Scholars and Innovative Research Team in University.
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.04.087.
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